Volume 70, Issue 7, Pages 1287-1295 (October 2006) Characterization of hyaluronan cable structure and function in renal proximal tubular epithelial cells  W. Selbi, C.A. de la Motte, V.C. Hascall, A.J. Day, T. Bowen, A.O. Phillips  Kidney International  Volume 70, Issue 7, Pages 1287-1295 (October 2006) DOI: 10.1038/sj.ki.5001760 Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 1 Expression of HA on HK2 cells. Confluent monolayers of HK2 cells were either (a) serum-deprived for 48h or (b) grown in the presence of 2% (v/v) FCS before fixation with methanol and detection of HA by addition of bHABP. Sections were imaged by confocal microscopy (× 10 objective). To confirm the nature of HA staining, in parallel experiments, cells were treated with (c) bovine testicular hyaluronidase (final concentration 200μg/ml) at 37°C for 5min, before fixation and addition of biotinylated HA-binding protein. Pericellular coats are highlighted by arrowheads and HA cables by white arrows. Original magnification × 10. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 2 Localization of IαI/PαI. HK2 cells were grown to confluence in the presence of 10% (v/v) FCS, before fixation with methanol. (a) HA was detected by bHABP and fluorescent avidin-D, and (b) IαI/PαI detected by addition of polyclonal antibody to IαI/PαI (dilution 1:100) and Alexa Flour 568 conjugate. (c) Colocalization of HA and IαI/PαI was examined by merging of individual images. HA cables are highlighted by arrows. Original magnification × 10. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 3 Incubation with antibody to IαI/PαI inhibits cable formation. Confluent monolayers of HK2 cells were incubated (a) in serum-free medium or serum-free medium to which polyclonal antibody to IαI/PαI had been added at a final dilution of either (b) 1:200 or (c) 1:50 for 24h before fixation with methanol. HA was detected using bHABP as before and sections imaged by confocal microscopy (× 10 objective). (d) In addition, confluent monolayers of cells were incubated with the polyclonal IαI/PαI antibody in the presence of 2% (v/v) FCS for 24h before fixation and visualization of HA. Pericellular coats are highlighted by arrowheads and HA cables by white arrows. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 4 Monocyte binding is decreased by inhibition of cable formation. Confluent monolayers of HK2 cells were incubated with polyclonal antibody to IαI/PαI or A38 monoclonal antibody to TSG-6 (5μg/ml) for 24h. Subsequently, the monolayer was washed with PBS before the addition of 1 × 10651Cr–labelled U937 cells again under serum-free conditions for 1h at 37°C. Quantitation of bound radioactivity was carried out as described in Materials and Methods. Data represent mean±s.d., n=3. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 5 Localization of versican. HK2 cells were grown to confluence in the presence of 10% (v/v) FCS, and serum deprived for 48h before fixation with methanol. (a) HA was detected by addition of bHABP and fluorescent avidin-D, and (b) versican detected by addition of monoclonal antibody to versican (clone 2B1) and Alexa Flour 568-conjugated antibody. (c) Colocalization of HA and versican was examined by merging of individual images. HA cables are highlighted by arrows. Original magnification × 10. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 6 HAS3 overexpression and characterization of HA. Confluent serum deprived monolayers of HAS3-overexpressing cells (open circles) or mock-transfected cells (boxes) were exposed to serum-free conditions for 24h in the presence of 20μCi/ml [3H]glucosamine. (a) Supernatant and (b) trypsin extracts were prepared as described in Materials and Methods. Radiolabelled HA was subsequently analyzed by Sephacryl S-500 chromatography. (c) Subsequently, the distribution of HA between high (>106Da), medium (105–106Da), and low (<105Da) molecular weight fraction for the mock-transfected and HAS3 cell lines were calculated and the data presented as the percentage of the total of HA in each molecular weight fraction. (d) In a parallel experiment, supernatant samples were collected from confluent serum-deprived mock-transfected and HAS3-overexpressing cells and HA quantified by enzyme-linked immunosorbent assay. In addition, the relative expression levels of HAS2 and HAS3 mRNA expression in the mock-transfected and HAS3-overexpressing cells were examined by reverse transcription-PCR. Total mRNA was extracted from confluent monolayers of mock-transfected and HAS3-overexpressing cells after 48h of serum deprivations. PCR products were separated on a 3% (w/v) agarose gel and stained with ethidium bromide. PCR amplification was carried out for 28 cycles for β-actin mRNA, 32 cycles for HAS2 mRNA, and 36 cycles for HAS3 mRNA. Densitometric ratios of the gene of interest compared to the housekeeping gene β-actin normalized to the ratio seen in the mock-transfected cells of three individual experiments are shown with the data representing mean±s.d. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 7 Confluent monolayers of HK2 cells, mock or HAS3-overexpressing cells were serum deprived for 48h before detachment. Cells were incubated with anti-CD44 common region antibody, and cell surface expression of CD44 was assessed by FACS analysis. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 8 Visualization of HA in HAS3-overexpressing cells. Confluent monolayers of (a) mock-transfected and (b) HAS3-overexpressing cells were serum deprived for 48h before fixation with methanol and detection of HA by the addition of bHABP. Sections were imaged by confocal microscopy (× 10 objective). Pericellular coats are highlighted by arrowheads and HA cables by white arrows. Original magnification × 20. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 9 Alterations in cell function associated with HAS3 overexpression. (a) Quantification of cell migration. Confluent serum-deprived monolayers of mock- (stippled bars) or HAS3-overexpressing cells (solid bars) were scratched as described in Materials and Methods to produce an intersecting area denuded of cells. In addition, for HAS3-overexpressing cells, to confirm the role of HA, following generation of a denuded area, cells were treated with bovine testicular hyaluronidase (final concentration 200μg/ml) at 37°C for 5min (cross-hatched bars). Subsequently, following washing of the monolayer to remove detached cells, the rate of cell migration of each was assessed by directly counting the number of cells migrating into the intersecting denuded area at each of the time points indicated. The data are expressed as the number of cells per mm2 of denuded area. Data represent the mean±s.d. of four individual experiments. (b) Quantitation of monocyte binding. Confluent monolayers of mock-transfected or HAS3-overexpressing cells were washed with PBS before the addition of 1 × 10651Ci-labelled U937 cells again under serum-free conditions for 1h at 37°C. Quantitation of bound radioactivity was carried out as described in Materials and Methods. To quantify HA-dependent binding, the monolayer of HAS3-overexpressing cells was treated with bovine testicular hyaluronidase (H'ase) (final concentration 200μg/ml) at 37°C for 5min before the addition of monocytes. Data represent mean±s.d. of six individual experiments. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions

Figure 10 Total mRNA was extracted from confluent monolayers of mock-transfected and HAS3-overexpressing cells after 48h of serum deprivation. PCR products were separated on a 3% (w/v) agarose gel and stained with ethidium bromide. For each gene of interest, three representative PCR reactions are shown. PCR amplification was carried out for 28 cycles for β-actin mRNA, 36 cycles for hyal1 mRNA, 32 cycles for hyal2 mRNA, 36 cycles for PαI HC3 mRNA, and 36 cycles for bikunin. Densitometric ratios of the gene of interest compared to the housekeeping gene β-actin of three individual experiments are shown with the data representing mean±s.d. Kidney International 2006 70, 1287-1295DOI: (10.1038/sj.ki.5001760) Copyright © 2006 International Society of Nephrology Terms and Conditions